TRPV1 is a non-selective cation channel crucially involved in transduction of nociceptive stimuli into pain signals. Consequently, inhibition of TRPV1 is one of the major strategies for designing next generation anti-pain drugs. One of the hallmarks of TRPV1 is its polymodal activation profile; that is, the ability to detect and, remarkably, integrate the information from diverse environmental factors (e.g. binding of ligands, pH and temperature) to initiate an action potential in the peripheral ends of sensory axons. From a molecular point of view, this polymodality is the result of the allosteric coupling between distinct sites acting as sensors for the diverse stimuli and the activation gate. This project investigates the mechanism of this allosteric coupling using computational biology (molecular dynamics, free energy calculations), state-of-the-art chemical biology (non-natural amino acids) and electrophysiology. Three fundamental questions raised by some of our recent research findings will guide our investigation. Aim 1 addresses the molecular mechanism of activation of TRPV1. The working hypothesis comes from our recently published computational work whose predictions have been, in part, already verified experimentally. We found that hydration and dehydration of four hydrophobic pockets present in the structure of TRPV1 affect the orientation of a conserved asparagine residue in S6; the rotation of this side chain is, in turn, responsible for the opening of the pore. We will test this hypothesis with an extensive set of calculations and experiments. Aim 2 Builds on the observation that wetting/dewetting phenomena show exquisite temperature dependence and thus provide a viable mechanism for heat activation. This aim is devoted to the quantitative characterization of the wetting/dewetting thermodynamics and to the experimental testing of our model using mutagenesis. Finally, Aim 3 investigates the mechanism underpinning TRPV1 regulation by PIP2. Our preliminary data suggest that this lipid favor a conformational transition of the pore lining S6 helix from a canonical ?-helix to a non-canonical conformation containing a segment of ?-helix. We will test this hypothesis using the combination of computational modeling, site directed mutagenesis, whole cell and excised patch electrophysiology.